The long term goal of this proposal is to generate novel inhibitors of fatty acid biosynthesis in Mycobacterium tuberculosis. It is hypothesized that such compounds will have antimycobacterial activity and will provide a appropriate starting point for generating drugs to treat multi-drug resistant tuberculosis. The proposal has two Aims. Specific Aim 1 focuses on the design and synthesis of inhibitors that target InhA, the enoyl reductase FASII enzyme. This enzyme is one of the putative targets for isoniazid, a frontline antituberculosis drug. Novel compounds will be synthesized based on the diphenyl ether skeleton of triclosan, an inhibitor of enoyl reductases in M. tuberculosis and other bacteria. Inhibitor design will utilize X-ray crystallography, Raman spectroscopy and computational approaches. Compounds will be tested using enzyme kinetics, the antimycobacterial activity will be assessed using MICs and the intracellular mode of action of the compounds will be evaluated using DNA microarrays and photoaffinity labeling. Specific Aim 2 will investigate the mechanism of action of isoniazid and will test the hypothesis that proteinprotein interactions within the mycobacterium modulate the sensitivity of InhA and other FASII enzymes to isoniazid. The FASII enzyme complex from M. tuberculosis will be purified and the activity and sensitivity of each enzyme component toward FAS inhibitors will be evaluated. Characterization of the FASII complex will reveal the identity of the dehydrase enzyme and the FASII complex will be reconstituted in vitro using recombinantly expressed proteins. In addition, pull-down experiments will be used to identify other InhA protein binding partners and characterization of the FASI enzyme complex from M. tuberculosis will be initiated.